The development of highly effective metal–zeolite bifunctional catalysts for the hydroisomerization of n-alkanes is a paramount strategy to produce second-generation biofuels with high quality. In this study, polyhexamethylene biguanide hydrochloride (PHMB) is precisely added to the initial gel to synthesize nanosized ZSM-23 zeolites (Z23-xPH). Due to orientation adsorption and steric hindrance effects of PHMB, each sample of Z23-xPH demonstrates enhanced mesoporosity in comparison with the conventional Z23-C zeolite. Furthermore, the Brønsted acid density of the Z23-xPH samples is also significantly reduced due to a reduction in the distribution of framework Al at T2–T5 sites. The corresponding Pd/23-C and Pd/Z23-xPH bifunctional catalysts with 0.5 wt% Pd loading for n-hexadecane hydroisomerization are prepared by incorporating ZSM-23 zeolites as acid supports. According to the catalytic test results, the suitable addition of PHMB can effectively promote the iso-hexadecane yield. The Pd/Z23-2PH catalyst with an nPHMB/nSi molar ratio of 0.002 demonstrates the highest maximum iso-hexadecane yield of 74.1% at an n-hexadecane conversion of 88.3%. Therefore, the employment of PHMB has provided a simple route for the development of highly effective Pd/ZSM-23 catalysts for n-alkane hydroisomerization.